Summary of Work: Mapping epitopes of the Human Immunodeficiency Virus (HIV) is important for the diagnosis of infection and for the development of vaccines and therapeutics for Acquired Immune Deficiency Syndrome (AIDS). We have been probing epitopes on the HIV proteins gp120 and p24. The initial step in the entry of HIV into the host cell is binding of the envelope glycoprotein gp120 to the cellular receptor CD4. Also gp120 elicits the major components of the protective immune response against HIV in humans and chimpanzees. gp120 and its synthetic peptides have been investigated as potential vaccine candidates. Similarly, HIV p24 elicits the first antibodies upon HIV infection. As the HIV infection progresses to AIDS, there is a simultaneous reduction in anti-p24 antibody titer. It has been proposed that a combination vaccine eliciting antibodies to both gp120 and p24 may be useful in combating HIV infection. Thus, knowledge of the antigenic determinants on p24 and gp120, especially those eliciting the formation of protective antibodies, is extremely important in the development of a vaccine. We have combined proteolytic footprinting and MALDI/MS to map epitopes on the native proteins recognized by antibodies. In this method, proteins affinity-bound to an immobilized antibody are proteolytically cleaved and the unbound fragments are removed by washing. The bound fragments containing the epitope are characterized by directly analyzing the immobilized antibody by MALDI/MS. We are currently mapping epitopes on gp120 recognized by human MAbs isolated from sera of HIV infected individuals. These huMAbs have been shown to neutralize laboratory strains of HIV in vitro, but often do not demonstrate strong neutralizing properties against primary isolates. Knowledge of the epitopes recognized on gp120 in combination with their neutralizing properties is necessary for vaccine development. In the case of one specific human MAB, 1331D, recognizing gp120, the epitope identified by ELISA was a simple linear epitope. We determined, however, that the MAb, in fact, recognized a discontinuous epitope on the native protein. these results also explain why two different human MAbs recognizing the C-terminus of gp120 map to the same core peptides by immunoflorescence show different clade specificities. We have also developed a methodology for improved sensitivity for use with impure antibodies based on initial use of an immobilized capture Ab that will capture the Ab of interest. The capture Ab is then crosslinked to the Ab of interest. Significantly reduced chemical background in the MALDI spectra leads to increased sensitivity.